MicroRNAs (miRNAs) are regulators of platelet function and may thus contribute to inter-individual variability in platelet reactivity. MiR-223-3p is the most abundant of the platelet-derived miRNAs. Several studies have reported an association between miR-223-3p levels and platelet reactivity or the recurrence of cardiovascular events, but this miRNA's impact on platelet function is still poorly understood. We aimed to investigate miR-223-3p's effects on platelet reactivity in platelets derived from human hematopoietic stem cells (CD34+) and to study the underlying mechanisms of its action. MiR-223-3p upregulation and downregulation were carried out by transfecting megakaryocytes (MKs) derived from CD34+ cells with a miR-223-3p mimic or Cas9/sgRNAs ribonucleoprotein complexes, respectively. Flow cytometry was used to quantify the expression of surface markers of MKs and platelets, platelet production, and platelet reactivity. Platelet-supported thrombin generation was quantified in human plasma. Downregulation of miR-223-3p resulted in fewer proplatelet swellings and decreased platelet production. MiR-223-3p upregulation and downregulation affected the proportion of procoagulant platelets. This phenotype was mirrored by changes in the gene expression of the transmembrane protein 16F (TMEM16F), a phospholipid scramblase that plays a key role in the generation of procoagulant platelets. A luciferase reporter gene assay validated that TMEM16F mRNA was a direct target of miR-223-3p. Platelet-supported thrombin generation was reduced when miR-223-3p was upregulated. In conclusion, miR-223-3p modulates the generation of procoagulant platelets.